1. Field of the Invention
This invention relates to the field of recombinant expression in insect cells. It relates especially to the expression of heterodimeric proteins in such cells and more particularly to the expression of glycoprotein hormones such as follicle stimulating hormone and the like.
2. State of the Art
Follicle stimulating hormone (FSH) belongs to the family of glycoprotein hormones, which are produced either in the pituitary (LH, TSH) or in the placenta (hCG). Within a species, each of these hormones consists of a common .alpha. subunit, which is non-covalently bound to a hormone specific .beta. subunit. Purified FSH administered alone or in combination with luteinizing hormone (LH), has been used to induce a superovulatory response. The results with these hormones or with pregnant mare serum gonadotropin (PMSG), which contains intrinsic FSH and LH activity, have been variable. The use of recombinant bovine FSH (rec.bFSH), which is guaranteed to be free of LH, and which is homologous to the species in which it is applied most frequently, may improve superovulation results. Furthermore, bovine FSH is difficult to purify in substantial quantities from bovine pituitaries (Wu et al., 1993). Rec.bFSH therefore may provide sufficient material to allow for structure-function studies by epitope mapping (Geysen et al., 1984; Westhoff et al., 1994).
cDNA's of bovine .alpha. subunit (Erwin et al., 1983; Nilson et al., 1983), as well as cDNA's of bovine FSH .beta. subunit (Esch et al., 1986; Maurer & Beck, 1986) have been isolated.
As indicated in Table 1, recombinant FSH has been produced in chinese hamster ovary (CHO) cells for the human (Keene et al., 1989; Van Wezenbeek et al., 1990; Roth et al., 1993) and the ovine (Mountford et al., 1994) species, whereas for the bovine species recombinant FSH has been produced in CHO cells and in transgenic mice (Greenberg et al., 1991). Rec.bFSH has also been produced in mouse epithelioid cells (Chappel et al., 1988) and has been applied for superovulation in cattle (Looney et al., 1988; Wilson et al., 1989, 1993).
The baculo virus expression system is based on the infection of insect cells with a recombinant baculovirus (L. A. King and R. D. Possee, 1992) and is increasingly used for production of heterologous proteins. Insect cells have the glycosylation apparatus capable of synthesis of high mannose or hybrid type carbohydrates, as well as simple O-linked chains, and recombinant proteins can be expressed with much higher efficiency as compared with the chinese hamster ovary or COS cell system (Chen et al., 1991). The baculovirus expression system has been used to produce amongst others the .alpha. subunit of hCG (Nakhai et al., 1991a,b), the .alpha. subunit of carp gonadotropin (Huang et al., 1991; Chen and Bahl, 1991), the .beta. subunit of hCG (Chen et al., 1991; Sridhar and Hasnain, 1993; Sridhar et al., 1993; Nakhai et al., 1992; Jha et al., 1992), hCG (Chen and Bahl, 1991; Nakhai et al., 1992), the receptor for human FSH (Christophe et al., 1993) and, quite recently, human FSH (Lindau-Shepard et al., 1994; Dias et al., 1994) (Table 1). Co-expression of two, or more, proteins by the baculovirus expression system has been achieved for instance by construction of a multiple expression transfer vector containing two, or more, foreign genes each of which is under the control of a copy of the p10 or polyhedrin promoter. Such expression vectors have been applied to the production of 2 totally unrelated proteins, for instance luciferase and hCG .beta. (Hasnain et al., 1994), but also to the production of 3 or 4 closely related proteins, which may be assembled in vivo to complex structures (Belayev and Roy, 1993). Such a system might also be used for co-expression of FSH .alpha. and FSH .beta., including the bovine forms. However, the synthesis of protein complexes has also been accomplished by co-infection of insect. cells with two different recombinant viruses. This has been applied to bluetongue virus proteins (French, Marshall & Roy, 1990), hCG (Chen & Bahl, 1991) and hFSH (Lindau-Shepard et al., 1994). Here we report for the first time the synthesis of bovine FSH in insect cells, by co-infection of cells with two recombinant viruses carrying the genes of bFSH.alpha. and bFSH.beta., respectively. This bFSH appears to be active in at least three different bioassay systems. Production in insect cells of only bFSH.alpha. was about 10 times higher than of only bFSH.beta., but co-infection of the two recombinant viruses resulted in production of heterodimer at a level comparable to that of bFSH.alpha. alone. A similar effect has been observed with the production of recombinant ovine FSH in Chinese hamster ovary cells (Mountford et al., 1994), and of recombinant hCG in monkey cells (Reddy et al., 1985).
TABLE 2 Production level.sup.1) (IU/ml for bioassays, and .mu.g/ml for ACA and specific activity.sup.2) (IU/.mu.g) of rbFSH batch assay 1/7/94 Y.sub.1 morphol.sup.3) 8.54 8.54 8.54 4.27 x .+-. S.D. 7.47 .+-. 2.14 S.A. 2.49 Y.sub.1 cAMP.sup.4) 19.1 29.9 23.9 x .+-. S.D 24.3 .+-. 5.41 S.A 8.1 Sertoli cell.sup.4) 13.7 4.4 2.7 x .+-. S.D. 6.90 .+-. 4.83 S.A. 2.3 OMI 15.0 31.0 x .+-. S.D. 23.0 .+-. 11.3 S.A. 7.7 ACA 1.8 1.6 5.6 x .+-. S.D. 3.0 .+-. 1.8 .sup.1) harvest at 72 hours after infection (p.i.), except when indicated ##EQU1## .sup.3) measurement of change in cell morphology .sup.4) measurement of cAMP (1/2 max. level), except when indicated.
Up to now no reports have been presented describing baculo expression of bovine FSH.
A surprising effect, obtainable by expressing bovine FSH in baculovirus based systems, is that very high biological activity is found, as demonstrated both in a heterologous system containing human FSH receptors, and in a homologous system containing bovine immature oocytes. It appears that the biological activity of baculo-derived rbFSH is at least as high as native FSH purified from pituitaries, or as rbFSH produced in higher eukaryotic cell systems.
This leads directly to an application in humans, especially in those cases in which administration of FSH needs to be carried out only a limited number of times, or in which the application can be carried out in vitro. Furthermore parts of the rbFSH molecule may act as an FSH antagonist and therefore can be used as a male contraceptive. This will only be possible if (fragments of) bovine FSH produced in baculovirus systems will not be immunogenic, and can therefore be used in humans without restrictions. Alternatively, bFSH or fragments of it may be used for vaccination against FSH as a means of contraception in the male. In the human this could be an attractive alternative for the use of hFSH, because a heterologous hormone (or part of it) may be more immunogenic than the homologous hormone.
For the bovine species the results of the oocyte maturation inhibition test lead to application in superovulation treatments in the bovine, where it can act as a substitute for Pregnant Mare Serum Gonadotropin (PMSG) or other hormones with FSH activity, in the treatment of reproductive problems such as anoestrus incomplete follicle development etc. It can also be used in in vitro experiments, for instance for the purpose of in vitro maturation and fertilization of oocytes. The biological activity of baculo-derived rbFSH in a rat-Sertoli-cell assay and a Y.sub.1 cell assay indicates that this biological activity most likely is not species specific. Applications therefore can be expected in other species than the human, bovine or rat, both in vivo and in vitro.
The invention further allows one to tailor the degree of sialylation, and thus the metabolic clearance rate and in vivo biological activity of FSH, by cloning the transsialydase-gene into the subunit-gene(s) containing baculo-vector. This may allow for addition of neuraminic acid to the glycan cores of rbFSH, and thus for increased biopotency.
Another part of the invention provides for fusion of (parts of) the bFSH.beta.- and bLSH.beta.-gene in order to tailor chimaeric hormones with a fixed ratio of FSH to LH bioactivity.
It will be understood that these kind of applications and embodiments lie within the scope of the present invention. Thus, where FSH is used in the present application this must be read as including fragments and/or derivatives thereof. It will also be clear that the exemplified vectors and/or regulatory elements are only examples and that other vectors capable of expression in insect cells will be suitable as well, as will other regulatory elements. The cloning techniques are also known in themselves and may be varied. The exemplified cell line is a well known and often used insect cell line. Other cell lines capable of being transfected by the vectors of the invention will also be applicable. Culture media for the transfected cells can be suitably selected by the person skilled in the art. Once bovine FSH has been expressed it is known how to isolate it from the culture. Once isolated and/or purified pharmaceutical preparations can easily be formulated using the knowledge obtained with other recombinant or isolated gonadotropins.
The invention will be explained in more detail in the following experimental part.